Abstract
Nine genes that encode proteins of the MAP65 family have been identified in the Arabidopsis thaliana genome. In this study, we reported that AtMAP65-2, a member of the AtMAP65 family, could strongly stabilize microtubules (MTs). Bacterially-expressed AtMAP65-2 fusion proteins induced the formation of large MT bundles in vitro. Although AtMAP65-2 showed little effect on MT assembly or nucleation, AtMAP65-2 greatly stabilized MTs that were subjected to low-temperature treatment in vitro. Analyses of truncated versions of AtMAP65-2 indicated that the region that encompassed amino acids 495–578, which formed a flexible extended loop, played a crucial role in the stabilization of MTs. Analysis of suspension-cultured Arabidopsis cells that expressed the AtMAP65-2-GFP fusion protein showed that AtMAP65-2 co-localized with MTs throughout the cell cycle. Cortical MTs that were decorated with AtMAP65-2-GFP were more resistant to the MT-disrupting drug propyzamide and to ice treatment in vivo. The results of this study demonstrate that AtMAP65-2 strongly stabilizes MTs and is involved in the regulation of MT organization and dynamics.
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Abbreviations
- EGTA:
-
Ethylene glycol-bis-(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid
- MAPs:
-
Microtubule-associated proteins
- MT:
-
Microtubule
- PIPES:
-
Piperazine-N,N′-bis (2-ethanesulfonic acid; 1,4-piperazinediethanesulfonic acid)
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Acknowledgements
We thank Dr. Bo Liu at UC Davis for critical reading of and comments on the manuscript. This research was supported by the National Key Basic Research Project of China (Grant 2006CB100101 to M. Y.) and National Natural Science Foundation of China (Grants 30421002, 30370707, and 30570925 to M. Y.; 30600312, and 30770186 to T. M.).
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H. Li and X. Zeng have contributed equally to this paper and are considered as joint first authors.
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Supplementary Fig. 1
Sequence alignment between the truncated versions of AtMAP65-1 and AtMAP65-2. a Sequence identity between AtMAP65-2 1-339 and AtMAP65-1 1–339 was ~83.78%. b Sequence identity between AtMAP65-2 340–578 and AtMAP65-1 340–587 was ~59.14%. (TIFF 13065 kb)
Supplementary Fig. 2
The truncated AtMAP65-2 proteins co-sediment with MTs. The His-AtMAP65-2 340–578, 1–339, 340–494, 495–578, and GST-AtMAP65-2 1–494 fusion proteins were incubated in the presence or absence of 4 μM MTs. After centrifugation, the supernatants and pellets were subject to SDS-PAGE analysis. Lanes 1 and 2, at a concentration of 0.5 μM, the truncated proteins bound to MTs. Lanes 3 and 4, at a concentration of 5 μM, the truncated proteins bound to MTs. Lanes 5 and 6, 5 μM fusion proteins in the absence of MTs. (TIFF 3339 kb)
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Li, H., Zeng, X., Liu, ZQ. et al. Arabidopsis microtubule-associated protein AtMAP65-2 acts as a microtubule stabilizer. Plant Mol Biol 69, 313–324 (2009). https://doi.org/10.1007/s11103-008-9426-1
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DOI: https://doi.org/10.1007/s11103-008-9426-1